Shuttling of Peroxisomal Targeting Sequence Receptors

描述（由申请人提供）：过氧化物酶体在脂质代谢中起作用。至少有25种人类疾病影响过氧化物酶体酶的功能或亚细胞位置，或损害过氧化物酶体的生物发生。蛋白质靶向到过氧化物酶体基质和膜是由过氧化物酶体靶向信号（PTSs）介导的，其中PTS1和PTS2允许蛋白质进入基质，而mPTS促进蛋白质进入膜。参与基质蛋白输入的两个关键过氧化物Pex5p和Pex7p分别结合细胞质中的PTS1和PTS2序列。在货物转运进入细胞器基质之前，所产生的货物受体复合物与过氧化物酶体膜上的其他过氧化物酶对接。关于Pex5p和Pex7p亚细胞位置的不同结果可以通过假设PTS受体在输入周期中从细胞质穿梭到过氧化物酶体基质，然后返回细胞质来调和。自上次提交以来，我们已经为人类Pex5p （HsPex5p）的扩展穿梭模型提供了证据。我们提出的工作重点是在人类和酵母细胞的输入周期中，Pex5p和Pex7p的行程，以及这个穿梭周期的各个步骤的机制细节。我们的研究将揭示这些受体的作用机制，这些受体在致命疾病中发生突变，并且相对于其他细胞器使用的信号序列受体是独特的。目标是：1。HsPex5p的哪些片段允许其进出过氧化物酶体？HsPex5p的不同同工异构体会穿梭吗？货物结合或HsPex5p与过氧化物酶体对接和转运机制的相互作用是其穿梭所必需的吗？Pex5p进出过氧化物酶体在酵母中是否保守？Pex5p转运到易位、膜易位、货物释放和受体输出等步骤的机制是什么？我们还将在体外重建受体穿梭。Pex5p进出过氧化物酶体需要哪些过氧化物酶？在酵母和哺乳动物中，Pex7p是否循环进出过氧化物酶体基质？如果是这样，那么Pex7p穿梭的机制是什么？

DESCRIPTION (provided by applicant): Peroxisomes play a role in lipid metabolism. At least 25 human diseases affect the function or subcellular location of peroxisomal enzymes, or impair peroxisome biogenesis. The targeting of proteins to the peroxisomal matrix and membrane is mediated by peroxisomal targeting signals (PTSs), of which PTS1 and PTS2 allow import of proteins to the matrix, and the mPTS facilitates import to the membrane. Two key peroxins involved in matrix protein import, Pex5p and Pex7p, bind the PTS1 and PTS2 sequences, respectively, in the cytosol. The resulting cargo-receptor complexes dock with other peroxins on the peroxisomal membrane, before cargo translocation occurs into the organelle matrix. The different results obtained regarding the subcellular locations of Pex5p and Pex7p can be reconciled by the hypothesis that PTS receptors shuttle from the cytosol to the peroxisome matrix and then return to the cytosol during the import cycle. We have provided evidence, since the last submission, for this extended-shuttle model for human Pex5p (HsPex5p). Our proposed work focuses on the itineraries of Pex5p and Pex7p during the import cycle in human and yeast cells, and the mechanistic details of various steps of this shuttling cycle. Our studies will reveal the mechanism of action of these receptors, which are mutated in fatal disorders and are unique relative to signal-sequence receptors used by other organelles. The aims are:1. What are the segments of HsPex5p that allow its import and export into and out of peroxisomes? Do different isoforms of HsPex5p shuttle? Are cargo binding or interactions of HsPex5p with the peroxisomal docking and translocation machinery necessary for its shuttling?2. Is the shuttling of Pex5p into and out of peroxisomes conserved in yeasts?3. What is the mechanism of steps in Pex5p shuttling such as transfer to the translocon, membrane translocation, cargo release and receptor export? We will also reconstitute receptor shuttling in vitro.4. What peroxins are necessary for the shuttling of Pex5p into and out of peroxisomes?5. Does Pex7p cycle into and out of the peroxisome matrix in yeast and in mammals? If so, what is the mechanism by which Pex7p shuttles?